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January 19, 2008

The Biochemical Basis of Celiac Disease

by Sam Savage

By Wieser, Herbert Koehler, Peter

ABSTRACT Celiac disease (CD) is an inflammatory disorder of the upper small intestine triggered by the ingestion of wheat, rye, barley, and possibly oat products. The clinical feature of CD is characterized by a flat intestinal mucosa with the absence of normal villi, resulting in a generalized malabsorption of nutrients. The prevalence of CD among Caucasians is now thought to be in a range of 1:100-300. There is a strong genetic association with human leukocyte antigens (HLA-)DQ2 and DQ8 and currently unknown non-HLA genes. During the last decade, intense biochemical studies have contributed to substantial progress in understanding the general principles that determine the pathogenesis of CD. The precipitating factors of toxic cereals are the storage proteins, termed gluten in the field of CD (gliadins and glutenins of wheat, secalins of rye, and hordeins of barley). There is still disagreement about the toxicity of oat avenins. The structural features unique to all CD toxic proteins are sequence domains rich in Gin and Pro. The high Pro content renders these proteins resistant to complete proteolytic digestion by gastrointestinal enzymes. Consequently, large Pro- and Gln-rich peptides are cumulated in the small intestine and reach the subepithelial lymphatic tissue. Depending on the amino acid sequences, these peptides can induce two different immune responses. The rapid innate response is characterized by the secretion of the cytokine interleukin-15 and the massive increase of intraepithelial lymphocytes. The slower adaptive response includes the binding of gluten peptides (native or partially deamidated by tissue transglutaminase) to HLA-DQ2 or -DQ8 of antigen presenting cells and the subsequent stimulation of T-cells accompanied by the release of proinflammatory cytokines such as interferon-gamma and the activation of matrix metalloproteinases. Both immune responses result in mucosal destruction and epithelial apoptosis. Additionally, stimulated T-cells activate B-cells that produce serum IgA and IgG antibodies against gluten proteins (antigen) and tissue transglutaminase (autoantigen). These antibodies can be used for noninvasive screening tests to diagnose CD. The current essential therapy of CD is a strict lifelong adherence to gluten-free diet. Dietetic gluten-free foods produced for CD patients underlie the regulations of the Codex Alimentarius Standard for Gluten-Free Foods. The "Draft Revised Codex Standard" edited in March 2006 proposes a maximum level of 20 mg of gluten/kg for naturally gluten- free foods (e.g., based on rice or corn flour) and 200 mg/kg for foods rendered gluten-free (e.g., wheat starch). Numerous analytical methods for gluten determination have been developed, mostly based on immunochemical assays, mass spectrometry, or polymerase chain reaction. So far, only two enzyme-linked immunosorbent assays have been successfully ring-tested and are commercially available. During the last decade, future strategies for prevention and treatment of CD have been proposed. They are based on the removal of toxic epitopes by enzymatic degradation or gene engineering and on blocking parts of the immune system. However, any alternative treatment should have a safety profile competitive with gluten-free diet.

Cereal Chem. 85(1):1-13

Celiac disease (CD), also known as celiac sprue and glutensensitive enteropathy, is one of the most frequent food intolerances worldwide. It may be defined as an inflammatory disease of the upper small intestine (duodenum, jejunum) in genetically susceptible individuals triggered by ingestion of wheat, rye, barley, and possibly oat products. The precipitating factors of CD are the storage proteins of these cereals, widely called gluten in the field of CD. The currently accepted theory of pathogenesis is based on a primary immune response to gluten proteins as antigens and to tissue transglutaminase (TG), which has been identified as an autoantigen. Classically, CD patients develop a flat intestinal mucosa with the absence of normal villi, a cellular infiltrate of the lamina propria and an increase in the number of intraepithelial lymphocytes (IEL) after ingestion of gluten. These characteristics may vary from patient to patient, depending on the severity and extent of disease. CD has been depicted as an iceberg, with the tip symbolizing overt CD, typified by the presence of clinical and histological manifestations (Logan 1992). A major part of the pictured iceberg represents silent CD (presence of lesions in the absence of symptoms) and latent CD (presence of antibodies to gluten and TG in the absence of intestinal injury and symptoms). Dermatitis herpetiformis, a blistering skin disease, is regarded as a variant of CD.

Previously, CD was considered to be a rare childhood disorder but it is actually a frequent condition present in every age. CD is mostly prevalent in Europe and countries to which Europeans have emigrated, including North America, South America, and Australia. However, CD is increasingly found in areas of the developing world such as North Africa, Middle East, and India. Several recent studies using serological screening followed by small intestinal biopsy have shown a prevalence of 1 in 100-300 individuals. The occurrence among first-degree relatives has been reported to be strongly increased (a 10%). Many different symptoms are associated with CD, which can be divided into intestinal features such as chronic diarrhea, steatorrhea, vomiting, and abdominal distension and into the results of malabsorption such as deficiencies of vitamins and minerals, failure to thrive, and loss of weight. It is likely only 10-15% of people with CD in Europe and North America are currently diagnosed. Untreated patients are at increased risk of anemia, edema, osteoporosis, infertility, T-cell lymphoma, and other malignancies. Numerous diseases are associated with CD such as insulin-dependent diabetes mellitus (prevalence of [asymptotically =]8%), thyroid disease (6-8%), and immunoglobulin A (IgA) deficiency (2-5%).

Noninvasive blood tests measuring antibodies to gluten and TG are a considerable aid for CD diagnosis and monitoring of the response to therapy but duodenal biopsy and tissue histology remain the gold standard. After diagnosis has been established, the lifelong strict withdrawal of gluten from the diet is the current essential treatment of CD. This means that products containing wheat, rye, barley, and possibly oats are not allowed with the exception of highly purified starches; the daily intake of gluten should not exceed 20 mg.

Since the discovery of Dicke (1950) that the ingestion of wheat was responsible for the symptoms of CD, numerous reviews on clinical and immunological features of CD appeared (for recent reviews see Ciclitira et al 2005; Koning et al 2005; Anderson and Wieser 2006; Brandtzaeg 2006; Jabri and Sollid 2006; Kagnoff 2007). In the present article, the biochemical aspects of CD are emphasized considering the structure of gluten proteins and its relation to CD, the pathomechanism, and the therapy.

THE PRECIPITATING FACTOR

Testing of Toxicity

Numerous in vivo and in vitro methods have been used to identify the CD toxicity of cereal proteins and peptides (reviewed by Troncone and Auricchio 1991; Shewry et al 1992). Most investigators would agree that in vivo testing is the gold standard for assessing toxicity. Initially, in vivo studies established toxicity by feeding tests mainly based on the production of symptoms such as steatorrhea or malabsorption of xylose. Later on, the technique of intestinal biopsy was introduced, which resulted in more precise conclusions on toxicity. However, most of these tests were not considered completely satisfactory because the optimal amount of gluten equivalents used to challenge patients and the duration of the challenge were uncertain. An additional impediment was that large amounts of protein (10-100 g of gluten equivalent) were necessary for challenging each patient, so that purified proteins and peptides could not be tested. The introduction of oral challenge by direct instillation into the small intestine, followed by biopsy after several hours, enabled the reduction of gluten equivalent to [asymptotically =]1 g. Changes in villus height, ratio of villus height to crypt depth, and the number of IEL were shown to be reliable parameters for toxicity assessment (Sturgess et al 1994; Fraser et al 2003; Dewar et al 2006).

The development of the in vitro systems for testing toxicity or immunogenicity opened a new era, particularly in the studies on peptides, as small amounts (1 mg equivalent of gluten or even less) could be tested. The organ culture of intestinal tissue of CD patients has been proposed to be the most reliable in vitro model. Tissue from patients with active CD is removed as part of the diagnostic procedure and incubated in a culture medium. The tissue shows improvement of enzyme activity and morphology in the medium alone, but not in the presence of CD toxic substances. More recently, T-cell lines and clones of cells from CD patients have been used to measure immunogenic effects of proteins and peptides. A frequently-used test is the T-cell proliferation assay performed by incubation of the putative antigen (a 100-200 pg/mL), antigen presenting cells (e.g., B-cells), CD specific T-cells, and tritiated thymidine. The proliferation of T-cells determined by scintillation measurement is a parameter for immunogenic effects. Additionally, the production of interferon (IFN)-y or interleukin (IL)-4 can be measured. However, it should be mentioned that Tcell lines and clones are frequently different in their reaction to antigens, and immunogenicity does not always correspond to toxicity demonstrated by in vivo or organ culture tests. A series of further in vitro assays has been used as screening tests for CD activity: organ culture tests with fetal rat or chicken intestine, leukocyte migration inhibition factor, macrophage proagulant activity, or agglutination of leukemia K562 cells. In any case, in vivo testing ultimately is necessary to evaluate CD toxicity. Toxic Cereals

Though the classical features of CD were clearly described more than a century ago, it was not until 1950 that the CD toxicity of wheat was established (Dicke 1950). Soon after, a series of investigations led to the conclusion that rye and barley were also harmful, whereas corn, rice, and buckwheat were not (reviewed by Shewry et al 1992; Kasarda 1994, 2001). There is still disagreement about the toxicity of oats. Early reports suggested that oat ingestion resulted in malabsorption of fat or xylose, whereas other studies found that moderate amounts of oats were not harmful. However, all these studies were based on a small number of patients and the challenge duration was brief. Moreover, oat samples were not tested for contamination with wheat, rye, or barley. New studies were more comprehensive, and a large number of CD patients were challenged with pure oats. It was reported that oats were well- tolerated clinically, did not cause histological changes, and did not induce immune responses (Janatuinen et al 1995; Srinivasan et al 1996; Hoffenberg et al 2000; Janatuinen et al 2002). In contrast, Lundin et al (2003) showed that a small number of CD patients developed partial villous atrophy and increased levels of IFN-gamma RNA after oat challenge. In another study, some CD patients had mucosal T-cells reactive to avenin, the storage protein fraction of oats (Arentz-Hansen et al 2004). Therefore, the inclusion of oats in the gluten-free diet is still debated.

Of the toxic or potentially toxic grains, only proteins from wheat and oats have been extensively studied for CD toxicity. Testing of rye and barley has been rather minimal, the strong similarities of their storage proteins with wheat gluten proteins, however, support their CD harmfulness. The taxonomy of plants might provide useful guidance in dividing grains into safe and unsafe (Kasarda 2001). All toxic grains (wheat, rye, barley) are found in a single tribe, the Triticeae, within the grass family (Poaceae). Considering this, all wheat species including bread and durum wheat, kamut, spelt, emmer, and einkorn or the wheat-rye crossbreed triticale have to be avoided by CD patients. Oats, remaining controversial regarding toxicity, belong to a separate tribe, the Aveneae. The nontoxic cereals including corn, sorghum, millet, and rice are still more distant from the Triticeae and show separated evolutionary lines within the grass family. Plants that did not fall in the grass family such as buckwheat, quinoa, and amaranth have been classified as safe.

Toxic Proteins

Structure. Traditionally, cereal storage proteins have been grouped into the soluble prolamin and the insoluble glutelin fractions according to their solubility in aqueous alcohols (Osborne 1924). Both fractions consist of numerous proteins; the prolamin fractions mainly contain monomelic proteins, and the glutelin fractions polymeric proteins are linked by interchain disulfide bonds. According to common structures, the storage proteins of CD toxic cereals have been classified into three groups: 1) a high molecular weight (HMW) group; 2) a medium molecular weight (MMW) group; and 3) a low molecular weight (LMW) group, the latter being the major group (Shewry and Tatham 1990; Wieser 1994). The proteins of these groups can be divided into different types on the basis of sequence homologies; structural data for representatives of these types are presented in Table I. The HMW group contains HMW glutenin subunits (HMW-GS) (wheat), HMW secalins (rye), and D-hordeins (barley). HMW-GS and HMW secalins can be subdivided into the jc- type and y-type. The molecular weights of the subunits have a range of 70,000-90,000. The amino acid compositions are characterized by high contents of Gin, Gly, and Pro, which together account for [asymptotically =]70% of total residues. They consist of three structural domains: 1) a nonrepetitive N-terminal domain of [asymptotically =]100 residues, 2) a nonrepetitive C-terminal domain with [asymptotically =]40 residues, and 3) a repetitive central domain of 400-700 residues. The central domain contains repetitive hexapeptides such QQPGQG (one letter codes for amino acids) as a backbone with inserted hexapeptides like YYPTSP and tripeptides like QQP or QPG. The nonrepetitive N- and C-terminal domains contain much less Gin, Gly, and Pro and more amino acid residues with charged side chains. In a native state, the proteins of the HMW group are aggregated through interchain disulfide bonds and are hardly extractable with aqueous alcohol.

The MMW group consists of the homologous col,2-gliadins (wheat), co-secalins (rye), C-hordeins (barley), and the unique co5gliadins (wheat). They have extremely unbalanced amino acid compositions characterized by high contents of Gin, Pro, and Phe, which together account for [asymptotically =]80% of total residues. Most regions of the amino acid sequences are composed of repetitive units like (Q)QPQQPFP or (Q)QQQFP. The proteins of the MMW group occur as monomers and are readily extractable with aqueous alcohol. The LMW group can be divided into monomelic proteins including alpha/beta- and y-gliadins (wheat), gamma-40k-secalins (rye), gammahordeins (barley), and avenins (oats), and into aggregated proteins including LMW-GS (wheat), gamma-75k-secalins (rye), and B-hordeins (barley). All these proteins have an N-terminal domain rich in Gin, Pro, and aromatic amino acids (Phe, Tyr) and a C-terminal domain with a more balanced amino acid composition and with most of the Cys residues. gamma-Gliadins, gamma-40k-secalins, and gamma-hordeins are homologous with frequent repetitive units like QPQQPFP. a/p- Gliadins are unique for wheat; their N-terminal domain is characterized by repetitive units such as QPQPFPPQQPYP. Avenins are the smallest proteins in the LMW group due to a shortened N- terminal domain with only three repetitive units (PFVQQQQ). The C- terminal domain is, in parts, homologous to those of alpha/betaand gamma-types, and, in parts, unique with Gin-rich repetitive sequences such as QPQLQQQVF. The N-terminal domain of LMW-GS is characterized by repetitive units like (Q)QQPPFS. gamma-75k- Secalins are homologous to gamma-40k-secalins, with the exception that the Nterminal domain is much longer. The B-hordeins are homologous to y-hordeins, with the exception that the positions of single Cys residues are different.

Toxicity. The search for the precipitating proteins of CD toxic cereals was mainly performed with wheat (reviews by Shewry et al 1992; Kasarda 1994; Wieser 1995). Fractionation of wheat flour and testing by feeding trials proved the toxicity of gluten that remains as a cohesive protein mass when wheat dough is washed to remove starch granules and other soluble constituents. Subsequent investigations on wheat gluten indicated that the alcoholsoluble prolamin fraction consisting of gliadins was the most toxic factor, whereas the effect of the insoluble glutelin fraction consisting of glutenins was described controversially as either nontoxic, weakly toxic, or as toxic as gliadins. Equivalent to the gliadin fraction of wheat, the prolamin fractions of rye (secalins) and barley (hordeins) were associated with CD toxicity, without serious testing however. Recent comparative studies on the intestinal immune response revealed that the gliadins, secalins, and hordeins elicited a similar increase in IFN-gamma production in cultured biopsies of CD patients (Bracken et al 2006). The amino acid composition of these three prolamin fractions was closely related to both CD toxicity and taxonomy of the cereals (Wieser et al 1980). Common characteristics were high Gin and Pro contents. The glutelin fractions of rye, barley, and oats have not been tested yet.

Both wheat gluten and gliadins have been involved in studies on the influence of structural changes on CD toxicity. Digestion with pepsin and trypsin alone or followed by pancreatin resulted in the retention of toxicity. Consequently, enzymatic hydrolyzates of gluten (e.g., Frazer's fraction III, a peptic tryptic digest) have been used frequently as positive controls for toxicity tests because of good solubility in water or salt solutions. The breakdown of disulfide bonds by oxidation or heating during the baking process did not destroy toxicity. Therefore, the three-dimensional structure of proteins is not important for the toxic effect. The complete degradation into free amino acids by strong HC1 hydrolysis, however, rendered gliadins harmless, as did extensive deamidation of Gin side chains by diluted HC1.

Hekkens et al (1970) were the first to establish the toxicity of a well-defined group of aggregable alpha/beta-type gliadins, called A-gliadin, by means of instillation into the small intestine, followed by biopsy. The toxicity of A-gliadin was subsequently confirmed by in vitro tests (e.g., organ culture tests). Later in vivo and in vitro studies indicated that all gliadin types (alpha/ beta-, gamma-, omega-type) contained the toxic factor (reviewed by Shewry et al 1992). However, a differentiation between omega5- and ω1,2-gliadins was not undertaken. Both types of wheat glutenins, HMW-GS and LMWGS, were not tested until recently. In vivo and in vitro tests revealed that HMW-GS exacerbate CD just as gliadins (Molberg et al 2003; Dewar et al 2006). T-cell stimulation tests on peptides from LMW-GS indicated that this protein type also has the potential to induce a CD-specific immune response (Vader et al 2002a). In contrast to wheat, the different storage protein types of rye (HMW-, omega-, gamma-40k-, gamma-75k-secalins) and barley (D- , C-, B-, gamma-hordeins) have not been tested up to now. Based on structural homologies with wheat proteins (Table I), it can be assumed that all of them are toxic for CD patients (Vader et al 2003). In conclusion, the entirety of storage proteins (prolamins + glutelins) of wheat, rye, barley, and possibly oats appears to be involved in activating CD. Toxic Peptides

Only a few attempts have been made to isolate pure peptides from toxic proteins and to test for CD toxicity (reviewed by Wieser 1995; Stern et al 2001). In agreement, those peptides active in organ culture tests were derived from the N-terminal region (positions 1- 55) of alpha/beta-gliadins (Table II). Peptides a(56-68) and alpha(247-266) did not show any toxic effect on intestinal tissue. The tetrapeptide sequences PSQQ and QQQP, which are common for active peptides were considered to be key sequences for further investigations (Wieser et al 1986; de Ritis et al 1988). Conformation studies showed that beta-turns were the dominant structural feature of active peptides (Tatham et al 1990).

Since 1987, the isolated peptides were replaced by synthetic peptides, which allowed in vivo tests in addition to in vitro tests due to the availability of relatively high amounts. Peptide alpha(206-217) instilled into the small intestine showed toxic effects in two CD patients in remission and no effect in the control group (Mantzaris and Jewell 1991). Sturgess et al (1994) performed in vivo challenge in four treated patients using three synthetic peptides from alpha/betagliadins. Peptide alpha(31-49) caused significant histological changes in four patients, whereas peptide alpha(202-220) generated minor damage in one of four patients, and peptide alpha(3-21) was inactive. Ala-substituted variants of peptide alpha(31-49) remained active in the organ culture test, when residues L31 and P36 were substituted, but lost toxicity when residues P38, P39, and P42 were substituted (Shidrawi et al 1995). In vivo testing of two patients performed by Marsh et al (1995) demonstrated the toxicity of peptides alpha(3143) and alpha(44-55); in contrast, peptide alpha(56-68) was not toxic. In vitro studies (organ culture test) on peptides alpha(31-43) and alpha(31-55) revealed CD toxicity at low concentrations, whereas peptide alpha(44- 55) was active only in high concentrations (Maiuri et al 1996). Four CD patients in remission underwent challenges with peptide alpha(56- 75) and a negative control peptide from beta-casein (Fraser et al 2003). The gliadin peptide caused intestinal damage in all the patients, while the casein peptide produced no response. In summary, the investigations described were, in parts, unsatisfactory with regard to the number of tests, purity of peptides, and accordance of results, but it could be concluded that most of the toxic sequences occur in the repetitive N-terminal domain of alpha/betagliadins and mainly consist of Gin, Pro, and aromatic amino acids (Phe, Tyr). Corresponding repetitive sequences of gamma-type and omega-type gliadins, secalins, and hordeins have not yet been tested by instillation or organ culture tests, but fit well into the potentially toxic sequences of a/p-gliadins.

Recently, the search for CD active peptides has shifted toward epitopes that stimulate T-cells of CD patients. A large number of synthetic peptides were derived from the alpha/beta- and gamma- gliadins, glutenins, secalins, hordeins, and avenins. The major part of the peptides was barely stimulatory in an untreated form but became stimulatory by partial deamidation after acid/heat or TG treatment. Table III presents a selection of T-cell stimulatory gluten peptides. Relations between peptide structure and immunogenicity are described below.

IMMUNOPATHOGENESIS

Genetics

Major histocompatibility complex (MHC) genes are the most important genetic determinants of predisposition to CD and they account for about half of the genetic effect (Sollid and lie 2005). CD has the strongest association with the human leukocyte antigen (HLA) class II alleles HLA-DQ2 and HLA-DQ8. The absence of HLA-DQ2/ 8 is a strong negative predictor of CD, and HLA typing can be helpful to exclude the diagnosis of CD. The fact that HLA-DQ2/8 are expressed in [asymptotically =]25% of healthy individuals, and the difference in concordance rates between monozygotic twins ([asymptotically =]80%) suggested the existence of CD specific non- HLA genes. The identification and characterization of these non-HLA genes is an ongoing challenging task. Several genome searches for risk factors have been performed. With the exception of HLA genes, there is relatively little consensus between the results, which indicate that each of the non-HLA genes has a relatively modest effect. Although there is likely to be a number of different genes involved in CD, it is possible that they vary in their contribution to disease susceptibility among different individuals, making them hard to identify (Kagnoff 2007). It is unknown, which additional nongenetic factors play a role in disease development, and the influence of currently unknown environmental factors such as infections cannot be ruled out. A recent prospective study provided indication that a high frequency of rotavirus infections may increase the risk of CD in childhood in genetically predisposed individuals (Stene et al 2006).

Usually food proteins are degraded into small peptides and amino acids by gastric, pancreatic, and brush-border enzymes. The high Pro content, however, renders CD toxic proteins highly resistant to complete proteolytic digestion. For example, the Prorich 33-mer peptide alpha2(56-88) (Table III) was shown by in vivo and in vitro studies in rats and humans, respectively, to resist all intestinal peptidases (Shan et al 2002). Relatively large fragments with high Pro and Gin contents are, therefore, cumulated in the small intestine. However, differences between CD patients who are in remission and healthy individuals in their ability to digest CD toxic proteins could not be detected. CD typical pathological events may start with an alteration of the barrier function of the intestinal mucosa. Infections, mechanical injuries, or chemical injuries have been proposed to impair mucosa integrity, whereby dietary gluten peptides reach the subepithelial lymphatic tissue (Schuppan and Hahn 2002). Recently, upregulation of zonulin, a protein that modulates intestinal permeability, was proposed to be responsible for the increased permeability in CD patients (Drago et al 2006). Furthermore, a pathogenetic function of enterocytes in CD has been suggested; morphological studies using immunofluorescence microscopy showed that increased amounts of gliadin peptides were present in intracellular compartments of enterocytes of CD patients in remission compared with control enterocytes (Friis et al 1992; Zimmer et al 1998). Gluten peptides that have passed the enterocyte layer and arrived at the lamina propria are able to trigger two immunological pathways; one is the adaptive immune response involving antigen presenting cells (APC), HLA-DQ2 or HLA-DQ8 heterodimers and CD4^sup +^ T-cells in the lamina propria, and the other presents the innate immune response thought to be a rapid effect on the epithelium (recently reviewed by Ciccocioppo et al 2005; Londei et al 2005; Hourigan 2006; Kagnoff2007).

HLA-DQ heterodimers. The adaptive immune system plays a central role in CD, which has the strongest HLA association of any MHC disease. Key events of the adaptive immune response are schematically shown in Fig. 1. CD is limited to genetically predisposed individuals with HLA-DQ2/8 heterodimers expressed on the cellular surface of APC (mature dendritic cells, macroaphages, or B- cells). However, DQ markers can also be found in [asymptotically =]25% of healthy individuals; thus, the presence of HLA-DQ2/8 is a necessary but not a sufficient precondition for CD. HLA-DQ2 is formed by an a-chain encoded by the gene HLA-DQ A 1*0501 and a P- chain encoded by HLA-DQ B 1*0201. HLA-DQ8 is formed by an a-chain encoded by HLA-DQ A 1*03/01 and a Pchain encoded by HLA-DQ B 1*0302. DQ2 and DQ8 chains had 91% of sequence identity for both chains (Constantini et al 2005). HLA-DQ2 is present in the vast majority of CD patients (9095%).

The molecular mechanism of the adaptive immune response is considered to involve the DQ2 and DQ8 molecules for binding gluten peptides in their peptide groove and presenting them to Tcells in the lamina propria. DQ2 and DQ8 are the HLA-class II molecules that bind peptides of variable length, usually with nine residues (pl- p9) and flanked by at least one or more additional residues, and prefer to bind peptides with a left-handed poly-Pro II helical conformation (Kagnoff 2007). Key anchor points in the groove of HLA- DQ2 are at positions pi, p4, p6, p7, and p9; negatively charged amino acids are preferred at p4, p6, and p7 and hydrophobic amino acids at pi and p9 (Table IV). Crystallography of HLA-DQ2 in complex with the immunogenic deamidated gliadin peptide QLQPFPQPELPY (a57- 68/E65) demonstrated the presence of an intricate hydrogen-bonding network between the two molecules (Kim et al 2004). The deamidated form of the peptide (E65) had a 25-fold higher affinity compared with the nondeamidated counterpart (Q65). The residue E65 was an important anchor residue (p6) because it participates in an excessive bond with Lys-beta71 of HLA-DQ2. Recent studies on various native and modified peptides from alpha/beta-gliadins and gamma- gliadins extended the knowledge basis for understanding the binding (Bergseng et al 2005; Qiao et al 2005). The computer modeling of HLA- DQ2 and its interaction with gluten peptides could distinguish which substitutions improve the binding and showed that Glu residues interact with specific positively charged amino acids of HLA-DQ2 (Constantini et al 2005). Rather less is known about the binding motif for HLA-DQ8. Functional binding studies suggested anchor positions pi and p9 are preferentially occupied by negatively charged residues and p4 by a hydrophobic residue. In agreement, the peptide-binding groove of both HLA-DQ2 and -DQ8 favors binding of peptides with negatively charged residues at key anchor positions. However, such negatively charged amino acids (Glu, Asp) are largely absent from CD toxic proteins and peptides generated in the gastrointestinal tract. This discrepancy was solved by discovery that tissue TG converts neutral Gin to negatively charged Glu. TABLE IV

Anchor Positions of DQ2-Restricted Epitopes from alpha/beta- and gamma-Gliadins"

Fig. 2. Protein/peptide modification by TG2. A, TG catalyzes cross-linking between the side chains of Gin and Lys. B, TG catalyzes deamidation of the side chain of Gin in the absence of primary amines.

Tissue transglutaminase. In 1997, tissue TG (TG2) was identified as the endomysial autoantigen in CD (Dieterich et al 1997). TG2 belongs to the ubiquitious family of calcium-dependent transamidating enzymes that catalyze the covalent cross-linking of a Gin residue (acyl-donor) with a Lys residue (acyl-acceptor), resulting in the formation of an epsilon-(y-glutamyl)-lysine isopeptide bond (Fig. 2A). Under certain conditions (e.g., when no e- amino group of Lys or other primary amino groups are available or the pH has been lowered), Gin is deamidated to Glu by reaction with water (Fig. 2B). The biological significance of the latter reaction has been established only in connection with CD. Sequencing of the TG2 gene in celiac and control populations did not detect differences associated with CD, however, the intestinal expression of TG2 in patients with CD is significantly increased. The molecular mass of TG2 is [asymptotically =]76,000 and three amino acid residues, Cys277, His335, and Asp358 form the active site of TG2 (Liu et al 2002). The enzyme is released from intracellular stores upon mechanical stress, inflammation, infection, or during apoptosis. Severe cell damage causes a calcium influx, which allows TG2 to cross-link several structural and functional intracellular proteins.

Molberg et al (1998) discovered that intestinal T-cells of CD patients recognize deamidated gluten peptides and it became possible to define gluten peptides with T-cell stimulatory activities. TG2 has specifity for only selected Gin residues, which depends on amino acids neighboring the target Gin. By means of synthetic peptide libraries, the relative positions of Pro and hydrophobic amino acids to Gin residues were crucial (Fleckenstein et al 2002; Vader et al 2002b). The sequences QXP, QXXF, and QQXF (X representing any amino acid and F representing the hydrophobic amino acid) were identified as preferred substrates for TG2, whereas the enzyme was not active on the QP or QXXP sequences. Based on these, TG2 recognition algorithms were designed and used to screen databases of various cereal proteins for privileged TG2 recognition sequences. Many similar epitopes were identified in wheat gliadins and glutenins, rye secalins, and barley hordeins (Vader et al 2002b). Oat avenins did not fit into these algorithms due to the low Pro content. This finding was confirmed by recent studies showing that TG2 increased the T-cell response to gliadins, secalins, and hordeins, but not to avenins (Kilmartin et al 2006).

Beside deamidation, TG2 autocatalyzes cross-links with gluten peptides and forms HMW complexes (Ciccocioppo et al 2003; Dieterich et al 2006). A potential physiological relevance for these complexes can be derived from the existence of autoantibodies that are directed against these cross-linked neo-epitopes (Dieterich et al 1997). Furthermore, TG2 catalyzed binding of gluten peptides to extracellular matrix proteins such as collagen. This haptenization and long-term immobilization of gluten peptides could be instrumental in the perpetuation of intestinal inflammation in CD (Dieterich et al 2006). The chemical characterization of complexes between TG2 and two immunodominant gliadin peptides (native a57-68/ Q65 and deamidated a57-68/E65) were reported by Fleckenstein et al (2004). Two types of covalent complexes were found: 1) the peptides were either linked through a thioester bond to the active site Cys of TG2 or 2) through isopeptide bonds to particular Lys residues of TG2. At a high molar excess of gliadin peptides to TG2, altogether six Lys residues of TG2 participated in isopeptide bond formation.

T-cell activation. After binding gluten peptides to the peptide groove of HLA-DQ2/8 on the cellular surface of APC, DQ/peptide complexes are presented to T-cell receptors (TCR) of glutensensitive CD4^sup +^ helper cells. Stimulatory peptides can differ from patient to patient. The minimum length of gluten peptides required for T-cell recognition is nine amino acid residues (Sollid 2002). Large peptides that contain multiple HLA-DQ binding epitopes have greater T-cell stimulatory activity than small peptides containing single binding sequence (Shan et al 2005). Deamidation of Gin by TG2 is not an absolute requirement for T-cell activation, in particular, early in the CD of children (Vader et al 2002a). The detailed mechanism, by which T-cells exert their damaging effect on the mucosa, is still unclear. Immunochemical studies demonstrated that the expression of cytokines IFN-gamma, IL-2, and tumor necrosis factor (TNF-)alpha is increased in the lamina propria with the subsequent release and activation of matrix metalloproteinases that cause degradation of extracellular matrix proteins followed by mucosal destruction and epithelial apoptosis (proinflammatory Thl response). Moreover, stimulated T-cells provide help for B-cells to produce IgA and IgG antibodies specific for gluten peptides, TG2, and complexes of peptides and TG2 (antiinflammatory Th2 response). It is likely that T- and B-cells recognize different parts of the peptide/TG2 complex, with T-cells reacting to the smaller gluten peptide and B-cells responding to the larger TG2 enzyme (Dewar et al 2004). The existence of a TG2/peptide complex provides an explanation as to why antibodies to TG2 are found only in patients who ingest gluten.

The diversity of stimulatory gluten peptides derived from wheat, rye, and barley is by far greater than was previously appreciated. A small assortment is presented in Table III. A common feature among immunogenic epitopes is the presence of multiple Gin and Pro residues, however several studies with modified peptides have revealed that other residues have also a strong influence on stimulatory effects. The sequence region 56-75 of alpha/beta- gliadins is one of the most frequently studied epitopes presented by HLA-DQ2 after deamidation of Gin at position 65 (Anderson et al 2000; Arentz-Hansen et al 2000; Ellis et al 2003). The sequence PQPE LPYPQPQLPY (a62-75/E65) was modified by Ala at each position except E65 and tested by T-cell stimulation (Ellis et al 2003). The results demonstrated that substitution of Q72, L73, P74, or Y75 had no effect on the stimulation index, whereas all the other substitutions abolished activity showing that each residue of PQPE LPYPQP contributed to T-cell stimulation (Fig. 3A). The authors postulated that residues Q63, P64, L66, P69, and P71 (anchor positions p2, p3, p5, and p8) may all interact with TCR and the others (pi, p4, p6, p7, p9) are key residues for DQ2 binding.

T-cell stimulatory gluten peptides important for DQ8 are distinct from those for DQ2. The HLA-DQ8 restricted epitope identified in HMW- GS 1 Dx2 had a minimum core region of residues 723-735 (QQGYYPTSPQQSG) and did not require deamidation for T-cell stimulation (van de Wal et al 1999). Residues Q724 and Q732 were expected to occupy the anchor positions pi and p9, respectively, within the DQ8/TCR complex. To identify the residues that contribute to T-cell activation, a series of substitution analogues was tested for T-cell activity (Moustakas et al 2000) (Fig. 3B). Any substitution at p2 (G725), p4 (Y727), p5 (P728), and p8 (P731) abrogated T-cell recognition. The critical role of P728 and P731 probably reflected the maintenance of the correct conformation of the peptide in the binding groove. However, in contrast to gliadin peptide analogues, substitutions were accepted at p3 (Y726), p6 (T729), and p7 (S730). Substitutions of Q724 (pi), Q733, or S734 had little effect, while substitution of Q732 (p9) by A or N reduced T- cell activity to 43 and 45%, respectively; p9 substitution of Q732 by charged residues (K or E) abolished activity. Taken together, T- cell recognition of some gluten peptides is highly dependent on the native sequence, whereas some flexibility in T-cell activation can be observed with other peptides. Humoral immunity. In untreated CD, specific serological antibodies to gliadin, reticulin, actin, endomysium, and TG are raised. Within the adaptive immune system, T- cells activated by gluten peptides elicit not only a proinflammatory Thl response, but also an antiinflammatory Th2 response, which promotes B-cell maturation and expansion of plasma cells that produce IgA and IgG serum antibodies (Schuppan et al 1998) (Fig. 1). The unanswered question is whether these antibodies play a role in pathogenesis of CD or represent a bystander phenomenon (Dewar et al 2004). Koning et al (2005) suggested that the antibodies are specific indicators of CD but they are not initiators of CD and are unlikely to cause disease symptoms. Serum antibodies can be used for noninvasive screening tests to diagnose CD and to control therapy. Previously, the determination of IgA antibodies to gliadin and endomysium was the most important test. In combination, they had positive and negative predictive values approaching 100%. The demonstration that the antigen for endomysium is tissue TG (Dieterich et al 1997) allowed the development of an ELISA for the detection of TG antibodies (Sulkanen et al 1998). This test is similar in sensivity and specificity to the endomysium antibody assay, but it is cheaper and the results are much easier to reproduce. However, after positive serological findings, an intestinal biopsy is necessary to confirm the diagnosis of CD.

Innate Immune System

Recent data suggest that certain toxic gluten peptides are not recognized by the adaptive immune system but stimulate the innate system characterized by the massive increase in IEL, one of the hallmarks of CD. IEL are phenotypically and functionally different from the lamina propria CD4^sup +^ gluten-specific T-cells. Two subsets of IEL bearing the apTCR or the gammadeltaTCR are linked to CD innate immunity. IL-15 has been considered a central player in this part of the gluten-induced immune response. The use of organ culture of small intestinal biopsies indeed revealed that neutralization of this cytokine might hamper the CD pathogenesis cascade (Maiuri et al 2003). IL-15 is produced both by epithelial and lamina propria cells in active CD and is not produced by Tcells and B-cells involved in the adaptive immune response. Gluten peptides such as alpha(31-43) (Table II) induce IL-15 secretion directly by the activation of enterocytes (Maiuri et al 1996), macrophages (Tuckova et al 2002), and dendritic cells (Nikulina et al 2004; Palova-Jelinkova et al 2005). These peptides do not bind to HLA-DQ2 or HLA-DQ8 and activate IL-15 production in CD patients but not in normal controls. The innate response is found in HLA-DQ2 positive CD patients but not in HLA-DQ2 positive nonceliac controls. This peculiar innate response may explain why only some of DQ2 positive individuals develop CD.

IL-15 stimulates IEL to express NKG2D receptor and epithelial cells to express MHC class I chain-related molecule A (MICA), the epithelial ligand of NKG2D (Huee et al 2004; Meresse et al 2004). Upon engagement of NKG2D receptor with MICA, IEL kill epithelial cells contributing to tissue destruction. A two-signal model is emerging for CD pathogenesis: signal 1 generated by innate immunity and signal 2 by adaptive immunity (Brandtzaeg 2006). The innate response may be necessary to initiate the adaptive response (Maiuri et al 2003). However, much more research remains to substantiate this approach to the complex pathogenesis of CD.

THERAPY

Gluten-Free Diet

After the diagnosis of CD has been established, permanent withdrawal of gluten from the diet is the current essential treatment. CD patients consume gluten-free foods from two different categories. First, they are allowed to eat a wide range of common products such as meat, fish, milk, fruits, and vegetables. In composite foods, however, it is difficult to recognize whether they are gluten-free or not. Moreover, CD patients should be aware of numerous foods that contain hidden sources of gluten such as thickened sauces and soups, pudding, or sausages. second, CD patients consume dietetic foods that are gluten-free according to the Codex Standard for Gluten-Free Foods adopted by the Codex Alimentarius Committee on Nutrition and Food for Special Dietary Uses. Dietetic gluten-free foods are mostly substitutes of products containing wheat, rye, and barley such as bread, other baked products, pasta, and beer. Common raw materials used for these products are corn, sorghum, rice, buckwheat, or chestnut, and common thickening agents are locust bean gum, guar gum, or methyl cellulose and various forms of it.

Gluten Analysis

Regulations. The Codex Standard for Gluten-Free Foods (Codex Stan 118, 1981) was established in 1981 and amended in 1983. Since, at that time, no method for measuring gluten was available, the nitrogen content set to 0.05% on a dry matter basis was the only methological point. In practice, this method was limited to the analysis of wheat starch used in the preparation of gluten-free foods. Kjeldahl and, more recently, Dumas procedures were used for nitrogen determination. A revision of this standard is now underway. According to the Codex Standard, gluten is defined as a protein fraction from wheat, rye, barley, oats, or crossbred cultivars, to which some persons are intolerant and that is insoluble in water and 0.5 mol/L of NaCl. The Draft Revised Standard (Codex document CL 2006/5, NFSDU 2006) proposes a gluten level of 20 mg/kg of dry mass for naturally gluten-free food and 200 mg/kg for food rendered gluten-free, such as wheat starch. For gluten analysis, prolamins should be extracted with 60% ethanol and quantitated by an appropriate method. The gluten content has to be calculated by multiplying the prolamin content by factor 2.

A number of analytical methods have been published to detect or determine gluten in foods. However, only a general outline of an analytical method has been given by the Draft Revised Standard because existing methods did not correspond to minimum requirements of sensitivity, selectivity, precision in repeatability and reproducibility, or they were not ring-tested and not available as commercial test kits. Moreover, problems arose with heated products such as bread and with partially hydrolyzed products such as malt products and beer. Three important steps should be . involved in the analytical procedure: 1) the complete extraction of CD toxic proteins; 2) the specific, accurate, and sensitive quantitation of these proteins; and 3) the calibration by means of a representative reference protein. For historical reasons, most analytical methods for gluten quantitation were based on the determination of CD toxic prolamins. The main methodical principles have been immunochemistry, mass spectrometry, and measurement of DNA.

Protein extraction. The first step of gluten analysis is the extraction of gluten proteins from raw material or food. According to the Draft Revised Standard, 60% ethanol is recommended for the extraction of prolamins. However, when the material to be analyzed has been heated, such as bread, the extractability of prolamins using aqueous alcohols is strongly reduced (Wieser et al 1998). Garcia et al (2005) demonstrated that the combination of aqueous alcohol with a reducing agent (2-mercaptoethanol) and a disaggregating agent (guanidine) the so-called cocktail, allowed the complete extraction of prolamins and reduced glutelins from both unheated and heat-processed foods. After diluting the extract, the cocktail did not affect the antibodies used for gluten determination.

Immunochemical methods. Immunoassays are based on the specific reaction of antibodies (immunoglobulins) produced by the immunization of animals (e.g., rabbits or mice) with antigens, the substance to be determined (CD toxic proteins or peptides). First attempts to identify wheat proteins in gluten-free products were based on the measurement of antigen-antibody precipitation by means of gel-diffusion techniques, immunoelectrophoresis, or countercurrent electrophoresis. To date, however, ELISA has been the most frequent method and recommended by the Draft Revised Standard. ELISA is based on labeling the detection antibodies or the antigen with an enzyme (e.g., horseradish peroxidase or alkaline phosphatase) and the sensitive reaction of the enzyme with an appropriate substrate that can be measured spectrophotometrically. The efficiency and limitation of ELISA methods developed until 1998 has been reviewed by Denery-Papini et al (1999). First, polyclonal antibodies (PAb), mostly raised against gliadins, were used. These PAb react with different binding sites (epitopes) of the antigens and the results are less influenced by cereal species or cultivar. A disadvantage is the high risk of cross-reactions with proteins from nontoxic grains and the poor reproducibility of antibody production.

Later on, monoclonal antibodies (MAb) produced after immunization by the fusion of isolated splenocytes with murine myeloma cells were applied in ELISA. MAb have tremendous advantages due to the absolute reproducibility of specificity and the ability to produce almost unlimited quantities. Two ELISA systems have been most frequently applied for gluten analysis: the sandwich ELISA and the competitive ELISA. The sandwich ELISA uses capture antibodies immobilized onto the microtiter plate and detection antibodies labeled with an enzyme. The antigen is sandwiched between capture and detection antibodies. This type of ELISA is suitable only for the detection of large antigens because the antigen must have at least two epitopes to bind both capture antibody and enzyme-labeled antibody. On the other hand, the competitive ELISA is suitable for the detection of small-sized antigens with only one epitope such as small peptides in partially hydrolyzed gluten-containing products. The unlabeled antibody is immobilized onto the microtiter plate and a limited and constant quantity of enzyme-labeled antigen together with the antigens from the samples are added. Both labeled and unlabeled antigens compete for the limited number of antibody binding sites. The greater the quantity of the sample antigen, the weaker the reaction of the enzyme-labeled antigen with the substrate. Numerous sandwich and competitive ELISA based on PAb or MAb have been developed in different laboratories and used for gluten determination, but only a few assays have been transferred to commercial test kits as being an important precondition for Codex regulations. Further preconditions are specificity (reaction with CD toxic proteins without cross-reaction with nontoxic proteins) and sensitivity (detection limit far below 20 mg of gluten/ kg). Skerritt and Hill (1990) developed a sandwich ELISA using MAb against heat-stable co-gliadins. Accordingly, this test has been suggested as suitable for quantifying gluten in all types of uncooked, cooked, and processed foods. A gliadin extract from the Australian cultivar Timgalen is provided as reference protein. Drawbacks of the assay are it recognizes prolamins from barley and oats very poorly and that the results are strongly cultivardependent due to different proportions of co-gliadins (Wieser et al 1994; Seilmeier and Wieser 2003). The assay has been ring-tested successfully, validated by the Association of Official Analytical Chemists (AOAC), patented, and marketed by several companies. Sensitivity of the different test kits indicated by the manufacturers is 20-160 mg of gluten/kg. A second Rapid Test Kit has been proposed to provide rapid qualitative or semiquantitative results and to be suitable for home use or in process quality control (Skerritt and Hill 1991). The Mendez group in Madrid developed a sandwich ELISA based on MAb (R5) raised against co- secalins and directed against epitopes like QQPFP, QQQFP, LQPFP, and QLPFP occurring in CD toxic sequences of prolamins (Valdes et al 2003). The assay is compatible with the cocktail extraction, has a detection limit of [asymptotically =]3 mg of gluten/kg, and is equally sensitive to wheat, rye, and barley prolamins, while cross- reactions with oat, corn, and rice proteins have not been observed. The R5 ELISA has been marketed and successfully ring-tested. In the mean time, different kits based on sandwich ELISA, competitive ELISA, and even sticks are on the market. In 2005, the R5 ELISA was endorsed as a type I method by the Codex Committee of Methods of Analysis and Sampling (CCMAS) and could serve as a basis for further Codex Standard regulations.

Mass spectrometry. In recent years, the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDITOF MS) has become a method for the determination of CD toxic prolamins (Mendez et al 1995). The sample preparation was quite easy. The procedure simply consisted of mixing prolamin extract with an detergent (octyl-beta-D-glucopyranoside) and an appropriate matrix (sinapinic acid) and an aliquot of this mixture is measured on a MALDI-TOF mass spectrometer. The comparison of CD toxic prolamins showed that gliadins, secalins, hordeins, and avenins had characteristic mass profiles that allowed the discrimination of cereal species (Camafeita et al 1998). The detection limit for gliadins was 0.01 mg/mL of extract.

The use of a reducing agent for prolamin extraction (e.g., the cocktail) was not a handicap for MS analysis. Altogether, MALDITOF MS is a highly valuable nonimmunological approach for detection and quantitation of gluten in foods. Its limitation lies in equipment so expensive that only a few spezialized laboratories are able to perform analyses.

Reference protein. Various reference proteins, mostly gliadins, have been produced by different laboratories and companies to establish a calibration curve for gluten determination. References were isolated from different sources of cereals and were minimally characterized as to protein content and composition. The results on gluten contents presented in literature were, therefore, difficult to judge. For this reason, the Draft Revised Standard recommends that a golden reference should be prepared by one laboratory under strictly standardized conditions. The European Prolamin Working Group (PWG) decided to organize the preparation of sufficient amounts of a reference gliadin (van Eckert et al 2006). Kernels of 28 representative European wheat cultivars were mixed and milled, the resulting flour was defatted and successively extracted with a salt solution and 60% ethanol. The alcoholic extract (gliadin) was purified by ultrafiltration, freeze-dried, and homogenized. The crude protein content (N x 5.7, Dumas) was 89.4% and the preparation contained 68% monomelic gliadins, 29% oligomeric HMW- gliadins, and 3% albumins and globulins. The product was homogenous and stable when it was stored at 37[degrees]C for 28 days. In summary, it meets all criteria important for a reference prolamin and is now distributed by the PWG for collective use.

Polymerase Chain Reaction. The group of Liithy in Berne, Switzerland, was the first to apply polymerase chain reaction (PCR) for gluten analysis. PCR is based on the determination of a specific DNA; in comparison with protein analysis, PCR is more sensitive by several orders of magnitude. PCR assay including a highly repetitive and specific genomic wheat DNA segment was used for the detection of wheat contamination in 35 different gluten-free food samples ranging from baking additives to heated and processed food samples (Allmann et al 1993). However, wheat starch with only a very low gluten content had a strong positive reaction and pure gluten used as additive could not be detected due to the lack of genomic DNA. Real- time PCR systems were developed for the quantitative determination of gluten contaminations (Sandberg et al 2003). The test allowed the discrimination of wheat, rye, barley, and oat contamination and gave a good correlation with ELISA results. In summary, the developed quantitative PCR systems were recommended as a highly sensitive tool for gluten analysis complementary to immunological methods. However, DNA from hydrolyzed products such as beer, syrup, and malt extract could not be detected by the PCR systems.

Alternative Therapies

The current treatment for CD is a lifelong strict gluten-free diet to prevent chronic enteropathy and reduce the risk of lymphoma or carcinoma. The dietary restriction, however, is a big challenge for CD patients, which may lead to poor compliance or inadvertent intake of gluten. There is, therefore, an urgent need to develop safe and effective therapeutic alternatives. Knowledge about the pathogenesis of CD has increased rapidly during the last decade and provides novel strategies for prevention and treatment of CD (reviewed by Sollid and Khosla 2005; Gianfrani et al 2006). Prorich peptides derived from digested gluten can survive the normal digestive process (Shan et al 2002). Supplementation with additional proteolytic enzymes (prolyl endopeptidases) may degrade CD toxic epitopes to avert an immune response. Peptidases from bacteria, fungi, and germinating cereals (wheat, rye, barley) have been proposed for therapy (De Angelis et al 2006; Hartmann et al 2006; Siegel et al 2006; Stepniak et al 2006). In addition, these peptidase have been recommended for decreasing the level of gluten proteins and peptides in a food before ingestion by CD patients.

Modification of the CD toxic grains by breeding or by genetic engineering has been proposed to create CD safe cereals. Some wheat cultivars lacked or contained only low amounts of CD toxic epitopes (Spaenij-Dekking et al 2005) and RNA interference might be a promising technique to silence genes encoding remaining harmful proteins (Becker et al 2007). Zonulin has been described as a key molecule for intestinal permeability (Drago et al 2006) and the use of an inhibitor of zonulin may have an important effect on the access of gluten peptides in the lamina propria. Another therapeutic strategy could be the inhibition of intestinal TG activity (Hausch et al 2003). However, this enzyme has a diverse biological role and even local inhibition may create some unpredicted adverse effects. Moreover, the discovery of TGindependent epitopes make this approach unlikely. There is ongoing interest in the use of altered peptide ligands (immunomodulation). By making specific point alterations in the sequence of CD toxic peptides, HLA binding affinity can be retained, but the T-cell response to those peptides may be downregulated. Treatment with an appropriate synthetic peptide may modulate immune response favorably. Some other blocking approaches have been suggested, for example, inhibition of the proinflammatory cytokines IFN-y and IL-15 or the inhibition of ligand-receptor interactions between MICA and NGK2D.

In summary, a series of studies has been done for alternative therapies of CD. Most CD patients, however, are effectively cured by gluten-free diet without any potential side effect. Any alternative treatment should have a safety profile competitive with the exclusion diet (Gianfrani et al 2006). One must carefully weigh the risks, benefits, and costs of alternatives and carefully define under what conditions and indications such alternative therapies might be warranted (Kagnoff 2007).

DISCUSSION AND FUTURE PROSPECTS

Though substantial progress has been achieved in understanding the general principles that determine the pathogenesis and treatment of CD, many key questions still remain unanswered. With respect to the precipating environmental factor, the entity of storage proteins from all species and crossbreeds of wheat, rye, and barley are now considered toxic for CD patients. The different types of these proteins are generally characterized by structural domains with sequences rich in Gin, Pro, and hydrophobic amino acids. Surprisingly, HMW subunits were also toxic, though they have sequences quite different from the other types (Table I). Thus, the spectrum of toxic epitopes has been considerably enlarged. Nevertheless, these epitopes are unique to CD toxic cereals and do not occur in other food proteins. A neverending question is that of oat toxicity, in that research and regulation could not reach any final decision during the last 50 years. Pro-rich parts of storage proteins resist gastrointestinal peptidases and are cumulated in the small intestine, but there is obviously no difference between CD and healthy individuals. Resistant peptides reach the lamina propria, but the exact mechanism of the epithelial passage and its putative pathology are still unknown and have to be clarified.

Gluten peptides are able to trigger two immunological pathways, the adaptive and the innate immune response. The adaptive response has been studied intensely during the last decade. In a first step, peptides, native or deamidated by TG, are specifically bound to HLA- DQ2 or HLA-DQ8 of APC. There is a strong genetic association between CD and HLA-DQ2/8, however 25% of healthy individuals express this HLA type. Therefore, it is urgently required to identify additional genetic and possibly environmental factors that contribute to CD activation. Tissue TG is strongly involved in the adaptive immune response. On the one hand, deamidation of Gin at specific positions within epitopes can considerably increase T-cell stimulation, even though deamidation is not an absolute requirement. On the other hand, TG forms complexes with gluten peptides and induces the secretion of serum antibodies to TG, which are valuable indicators of CD. The role of these antibodies in CD pathogenesis, however, is unclear. After binding to HLADQ2/8, gluten peptides are presented to TCR, resulting in the secretion of proinflammatory cytokines, activation of matrix metalloproteinases, and subsequent destruction of mucosa. Certain gluten peptides, however, are not recognized by the adaptive response; they activate the innate response characterized by the secretion of IL-15 and the massive increase of IELS, which kills the epithelial cells. This new approach led to questions about whether the adaptive and the innate immune responses exist independently or whether they both are required to induce mucosal atrophy. Much more research work remains to find answers to these questions.

A strict withdrawal of gluten from the diet is an effective therapy for most CD patients and all prospective alternative therapies proposed in the literature must be weighed carefully with respect to risks, benefits, and costs. The success of the classical gluten exclusion therapy should be assured by gluten-free foods specifically produced for CD patients. Reliable methods for the quantitative determination of gluten in gluten-free foods are essential for CD patients, the food industry, and food control. However, the comparatively slow progress has been made in developing methods since the recognition that wheat gluten exacerbates CD (1950). Approximately 30 years passed until the first Codex Standard for Gluten-Free Foods was established in 1981, presenting a method that was limited to the analysis of wheat starch. Since that time, a revision of this standard is underway but a definite decision on disputed points (allowed gluten level, oat toxicity, analytical method) appears to be a distant prospect. ELISA has been the most frequently used method and different kits are currently on the market. In accordance with the Draft Revised Codex Standard, the kits contain antibodies only against prolamins but not against glutelin subunits, and the reference protein is a gliadin preparation. The gluten content has to be calculated by multiplying the prolamin content by a factor of 2. This calculation is invalid because the ratio of prolamins to glutelins is strongly dependent on cereal species and cultivars and is different in products derived from cereals. Therefore, a future task will be the development of a method for the determination of all types of storage proteins from wheat, rye, barley, and possibly oats, and the preparation of corresponding reference proteins.